The broad aim of this project is to understand at the cellular and molecular level the mechanisms involved in regulation of calcium entry across the plasma membrane in non-excitable cells. This is a process that is ubiquitous to non-excitable cells, occurs in many excitable cell types and which is known to play an important role in the control of a large variety of cell processes, including secretion, contraction, motility, growth, differentiation and apoptosis. This signaling process is potentially vulnerable to environmental intervention by chemical and physical agents (such as EMF). Capacitative calcium entry is believed to involve a signal generated in the endoplasmic reticulum when Ca2+ is released by IP3 and which then interacts with the plasma membrane to activate calcium channels. The nature of this signal and the nature of the channels involved are prime areas of investigation. Specifically, we are intested in the roles of Stim1 and Stim2, which act as sensors for endoplasmic reticulum calcium, and Orai1, 2 and 3, which function as subunits of the store-operated channels. We utilize as a model for studying this process the actions of a tumor-promoting plant product, thapsigargin. Thapsigargin acts by inhibiting endoplasmic reticulum Ca2+ pumps, thereby depleting intracellular Ca2+ stores in a passive manner. We utilize the tecnhiques of single cell fluorescence imaging, confocal microscopy, total internal reflectance microscopy and patch-clamp electrophysiology. We are currently using mouse models to evaluate the role of this pathway in various physiological processes, including bone homeostasis, skin development, wound healing, cell migration and chemotaxis. We are also interested in other calcium-permeable channels activated as a result of surface receptor activation, specifically the phospholipase C-regulated TRPC channels. There are 7 members of the TRPC family of ion channels identified in mammalian cells. In humans, there are 6, as TRPC2 is a pseudogene. These fall into three categories based on sequence similarity: TRPC1, TRPC3/6/7 and TRPC4/5. We are expressing these channel genes in cell types in which they are normally expressed and in cell types which do not express these channel proteins. We intend to examine the effects of interfering with, or deletion of these proteins by transfection of cells with siRNAs, and cDNAs encoding for potentially dominant negative peptides. We are currently investigating the mechanisms by which lipids regulate this class of channels, specifically, diacylglycerols and phosphatidylinositol 4,5-bisphosphate. We are hopeful that by achieving a better understanding of the molecular and cellular modes of regulation of these important signaling pathways, we can learn how calcium entry is altered by environmental factors and by disease states.